A biological indicator is a standardized preparation of viable microorganisms, usually bacterial spores, that is carried either directly by some of the items to be sterilized or by carriers such as filter papers, porcelain cylinders, that serve as a challenge to the effectiveness of a given sterilization cycle

1. Presented By: DHRUTI AVLANI
Roll No.: 27720316018
Reg No.: 162772310024
M.Pharm, 1st
Year, 2nd
Semester
NSHM Knowledge Campus, Kolkata – Group of Institutions
GUIDE: Dr. Sutapa Biswas Majee

2. VALIDATION is defined as a documented procedure for recording &
interpreting data required to show that any process (machine/method) will
consistently comply with predetermined specifications
Sterilization refers to the complete absence or destruction of all micro-organisms
from a surface, equipment, medications (parenterals or ophthalmics), or
biological culture medium.
Various methods of sterilization include thermal methods (dry or moist heat) and
non-thermal methods (physical – filtration or radiation and chemical - gaseous).
Validation of the process of sterilization is necessary to ensure
that sterilization conditions can keep the bioburden (load of
specific micro-organisms or their spores) to the desired level i.e.
to assure desired level of sterility, to maintain batch to batch
consistency with respect to sterility, to reduce rejection, to
ENSURE SAFETY.
Validation of the process of sterilization is necessary to ensure
that sterilization conditions can keep the bioburden (load of
specific micro-organisms or their spores) to the desired level i.e.
to assure desired level of sterility, to maintain batch to batch
consistency with respect to sterility, to reduce rejection, to
ENSURE SAFETY.

3. Therefore, to be operated at conditions less than the conditions
stated in the sterilization process specifications.
Aseptic
manufacture
of products
(106
) in
suitable
container-
closure
system
Site Non-
Sterile
Non-
Sterile
Non-
Sterile
REJECT
REJECT
ACCEPTED
DESIRED SAL
Container
Solution
Closure
Microbial
Challenge
Test
105
103
1
IDENTIFICATION
OF STERILIZATION
CYCLE
PARAMETERS
IN-PROCESS VALIDATION NECESSARY
Monitoring
sterility level

5. • Standardized preparation of viable microorganisms (bacterial spores), of
known sterilization resistance to the sterilization mode.
•Microbial preparations are carried either directly by some of the items to be
sterilized or by carriers such as filter papers, porcelain cylinders.
•Some biological indicators may also contain two different species and
concentrations of microorganisms
•Biological indicators (BI) therefore, serve as a challenge to the effectiveness
of a given sterilization cycle.
P
U
R
P
O
S
E

7. The stock suspension should contain predominantly dormant
(non-germinating) spores that are held in a non-nutritive liquid.
The finished product (microbial suspension, inoculated carriers,
or biological indicators) supplied by commercial manufacturers
shall have no microorganisms, other than the test
microorganism, present in sufficient numbers to adversely affect
the product.
The system to minimise the presence of microorganisms other
than the biological indicator microorganism in the product will
itself also be validated, monitored, and recorded.

8. Biological indicator survival is predicated upon both resistance and
population. Therefore, a 106
biological indicator population is not
always required to demonstrate a 106
SAL.
The appropriate use for biological indicators is to employ them
to confirm that the developed process parameters result in the
desired SAL.
Parameters used for thermal calculations and to
define the rate of thermal lethality:
D - Value and Z - Value

9.  ↑ the temperature (hot) ↑ the microorganisms die --- FASTER
PROCESS
 The speed of the process is expressed with the D-Value
 D-value refers to decimal reduction time
D-VALUE is the exposition time required (in minutes) to kill 1 log
(90%) of the microorganisms.
Thus, after an organism is reduced by 1 D, only 10% of the
original organisms remain.
D-VALUE is the exposition time required (in minutes) to kill 1 log
(90%) of the microorganisms.
Thus, after an organism is reduced by 1 D, only 10% of the
original organisms remain.
 For example: A hypothetical organism is reduced by 90% after exposure
to temperatures of 300 degrees Fahrenheit for 2 minutes, thus the D-value
would be written as D300F = 2 minutes. If the initial population was
100cfu/ml, then 10cfu/ml would remain after a log cycle reduction.
The decimal reduction time is dependent on the temperature, the type of
microorganism and the composition of the medium containing the
microorganism

10. D-value
t1= Initial Time t2= Final Time
N0= Initial Population N1= Final Population
Therefore, from the graph-
D121 = 45 – 30 / 5 - 4
D121 = 15/1
D121 = 15 sec
LogSurvivors
Time (min) at 121°C
DT =
t2 - t1
log N0 - log N1
Biological indicators with substantive D values and populations substantially
less than 106
are adequate to validate sterilization and decontamination
processes.
For example : Bacillus stearothermophilus spores on a strip has a normal
population of 1.4 * 105
and a D value of 1.5 min at 121°C.

11. It is the measure of change in death
rate with a change in temperature.
The Z-Value gives an indication of the
relative impact of different
temperatures on a microorganism,
with smaller values indicating greater
sensitivity to increasing heat.
Z =
t2 - t1
log a - log b
Z-value
t1= Initial Temperature a= Upper D-value
t2 = Final Temperature b=Lower D-value
Therefore, from the graph-
Z = 240 – 220 / log 100 – log
10
Z = 20 / 2 – 1
ThermalDeathTime(min)(D-value)
Temperature (°F)

13. PRODUCTASPETIC CESSING
TERMINAL
STERILIZATION
FINAL
PACKAGE
PRO
For sterile preparations (parenterals and ophthalmics), it is often necessary to
reach a compromise between the most effective sterilization procedure that will
kill microoragnisms and the one that will not have any adverse effect upon the
material to be sterilised.
Validation of sterilization process with biological indicator will ensure that
only the most effective sterilization procedure will ensure sterility of raw
materials to be used in aseptic processing and also terminally sterilised finished
products with desired sterilization assurance level.

14. 1) U.S. Pharmacopoeia-National Formulary [USP 32 NF 27]. Roydan,
Md: United States Pharmacopeial Convention, Inc; 2009. [1035]
Biological indicators for sterilization; 1: 416.
2) N.K.Jain; Sterilization. Pharmaceutical Microbiology; 2nd
edition,
7th
reprint; page no. 100-113; Vallabh Prakarshan, Delhi; 2013.
3) Roop.K.Khar, SP Vyas, Farhan J Ahmad, Gaurav K Jain;
Sterlization. Lachman/Lieberman’s The Theory and Practise of
Industrial Pharmacy; 4th edition; page no. 804-827; CBS
Publications & Distributors Pvt. Ltd., New Delhi, 2013.